Julia P. Steringer

PI(4,5)P2 Dependent Oligomerization of Fibroblast Growth Factor 2 (FGF2) Triggers the Formation of a Lipidic Membrane Pore Implicated in Unconventional Secretion

Background: PI(4,5)P2- and tyrosine phosphorylation-dependent unconventional secretion of FGF2 is mediated by direct translocation across the plasma membrane. Results: PI(4,5)P2-mediated membrane recruitment causes oligomerization of tyrosine-phosphorylated FGF2 that, in turn, triggers the formation of a lipidic membrane pore. Conclusion: Membrane-inserted FGF2 oligomers represent intermediates of membrane translocation during unconventional secretion. Significance: Mechanistic insight into a novel self-sustained mechanism of protein translocation across membranes is provided. Fibroblast growth factor 2 (FGF2) is a critical mitogen with a central role in specific steps of tumor-induced angiogenesis. It is known to be secreted by unconventional means bypassing the endoplasmic reticulum/Golgi-dependent secretory pathway. However, the mechanism of FGF2 membrane translocation into the extracellular space has remained elusive. Here, we show that phosphatidylinositol 4,5-bisphosphate-dependent membrane recruitment causes FGF2 to oligomerize, which in turn triggers the formation of a lipidic membrane pore with a putative toroidal structure. This process is strongly up-regulated by tyrosine phosphorylation of FGF2. Our findings explain key requirements of FGF2 secretion from living cells and suggest a novel self-sustained mechanism of protein translocation across membranes with a lipidic membrane pore being a transient translocation intermediate.

The Startling Properties of Fibroblast Growth Factor 2: How to Exit Mammalian Cells Without a Signal Peptide at Hand?

For a long time, protein transport into the extracellular space was believed to strictly depend on signal peptide-mediated translocation into the lumen of the endoplasmic reticulum. More recently, this view has been challenged, and the molecular mechanisms of unconventional secretory processes are beginning to emerge. Here, we focus on unconventional secretion of fibroblast growth factor 2 (FGF2), a secretory mechanism that is based upon direct protein translocation across plasma membranes. Through a combination of genome-wide RNAi screening approaches and biochemical reconstitution experiments, the basic machinery of FGF2 secretion was identified and validated. This includes the integral membrane protein ATP1A1, the phosphoinositide phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), and Tec kinase, as well as membrane-proximal heparan sulfate proteoglycans on cell surfaces. Hallmarks of unconventional secretion of FGF2 are: (i) sequential molecular interactions with the inner leaflet along with Tec kinase-dependent tyrosine phosphorylation of FGF2, (ii) PI(4,5)P2-dependent oligomerization and membrane pore formation, and (iii) extracellular trapping of FGF2 mediated by heparan sulfate proteoglycans on cell surfaces. Here, we discuss new developments regarding this process including the mechanism of FGF2 oligomerization during membrane pore formation, the functional role of ATP1A1 in FGF2 secretion, and the possibility that other proteins secreted by unconventional means make use of a similar mechanism to reach the extracellular space. Furthermore, given the prominent role of extracellular FGF2 in tumor-induced angiogenesis, we will discuss possibilities to develop highly specific inhibitors of FGF2 secretion, a novel approach that may yield lead compounds with a high potential to develop into anti-cancer drugs.

HIV-Tat Protein Forms Phosphoinositide-dependent Membrane Pores Implicated in Unconventional Protein Secretion

Background: Unconventional secretion of both HIV-Tat and FGF2 depends on the phosphoinositide PI(4,5)P2. Results: HIV-Tat forms membrane-inserted oligomers concomitant with PI(4,5)P2-dependent membrane pore formation. Conclusion: HIV-Tat and FGF2 show similar properties with a tight correlation between membrane pore formation and unconventional secretion from cells. Significance: Evidence is provided that suggests a common mechanism of unconventional secretion with potential relevance for a broad range of cargoes. HIV-Tat has been demonstrated to be secreted from cells in a phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-dependent manner. Here we show that HIV-Tat forms membrane-inserted oligomers, a process that is accompanied by changes in secondary structure with a strong increase in antiparallel β sheet content. Intriguingly, oligomerization of HIV-Tat on membrane surfaces leads to the formation of membrane pores, as demonstrated by physical membrane passage of small fluorescent tracer molecules. Although membrane binding of HIV-Tat did not strictly depend on PI(4,5)P2 but, rather, was mediated by a range of acidic membrane lipids, a functional interaction between PI(4,5)P2 and HIV-Tat was critically required for efficient membrane pore formation by HIV-Tat oligomers. These properties are strikingly similar to what has been reported previously for fibroblast growth factor 2 (FGF2), providing strong evidence of a common core mechanism of unconventional secretion shared by HIV-Tat and fibroblast growth factor 2.

Formation of disulfide bridges drives oligomerization, membrane pore formation, and translocation of fibroblast growth factor 2 to cell surfaces

Background: FGF2 translocation across plasma membranes depends on phosphoinositide-dependent oligomerization and membrane pore formation. Results: Two unique surface cysteines are critical for efficient FGF2 oligomerization, membrane pore formation, and FGF2 secretion from cells. Conclusion: Formation of intermolecular disulfide bridges drives phosphoinositide-dependent FGF2 oligomerization at plasma membranes. Significance: A new cis element critical for unconventional secretion of FGF2 was identified and validated. Fibroblast growth factor 2 (FGF2) is a key signaling molecule in tumor-induced angiogenesis. FGF2 is secreted by an unconventional secretory mechanism that involves phosphatidylinositol 4,5-bisphosphate-dependent insertion of FGF2 oligomers into the plasma membrane. This process is regulated by Tec kinase-mediated tyrosine phosphorylation of FGF2. Molecular interactions driving FGF2 monomers into membrane-inserted FGF2 oligomers are unknown. Here we identify two surface cysteines that are critical for efficient unconventional secretion of FGF2. They represent unique features of FGF2 as they are absent from all signal-peptide-containing members of the FGF protein family. We show that phosphatidylinositol 4,5-bisphosphate-dependent FGF2 oligomerization concomitant with the generation of membrane pores depends on FGF2 surface cysteines as either chemical alkylation or substitution with alanines impairs these processes. We further demonstrate that the FGF2 variant forms lacking the two surface cysteines are not secreted from cells. These findings were corroborated by experiments redirecting a signal-peptide-containing FGF family member from the endoplasmic reticulum/Golgi-dependent secretory pathway into the unconventional secretory pathway of FGF2. Cis elements known to be required for unconventional secretion of FGF2, including the two surface cysteines, were transplanted into a variant form of FGF4 without signal peptide. The resulting FGF4/2 hybrid protein was secreted by unconventional means. We propose that the formation of disulfide bridges drives membrane insertion of FGF2 oligomers as intermediates in unconventional secretion of FGF2.

Key steps in unconventional secretion of fibroblast growth factor 2 reconstituted with purified components

FGF2 is secreted from cells by an unconventional secretory pathway. This process is mediated by direct translocation across the plasma membrane. Here, we define the minimal molecular machinery required for FGF2 membrane translocation in a fully reconstituted inside-out vesicle system. FGF2 membrane translocation is thermodynamically driven by PI(4,5)P2-induced membrane insertion of FGF2 oligomers. The latter serve as dynamic translocation intermediates of FGF2 with a subunit number in the range of 8-12 FGF2 molecules. Vectorial translocation of FGF2 across the membrane is governed by sequential and mutually exclusive interactions with PI(4,5)P2 and heparan sulfates on opposing sides of the membrane. Based on atomistic molecular dynamics simulations, we propose a mechanism that drives PI(4,5)P2 dependent oligomerization of FGF2. Our combined findings establish a novel type of self-sustained protein translocation across membranes revealing the molecular basis of the unconventional secretory pathway of FGF2. DOI: http://dx.doi.org/10.7554/eLife.28985.001

Small Molecule Inhibitors Targeting Tec Kinase Block Unconventional Secretion of Fibroblast Growth Factor 2.

Fibroblast growth factor 2 (FGF2) is a potent mitogen promoting both tumor cell survival and tumor-induced angiogenesis. It is secreted by an unconventional secretory mechanism that is based upon direct translocation across the plasma membrane. Key steps of this process are (i) phosphoinositide-dependent membrane recruitment, (ii) FGF2 oligomerization and membrane pore formation, and (iii) extracellular trapping mediated by membrane-proximal heparan sulfate proteoglycans. Efficient secretion of FGF2 is supported by Tec kinase that stimulates membrane pore formation based upon tyrosine phosphorylation of FGF2. Here, we report the biochemical characterization of the direct interaction between FGF2 and Tec kinase as well as the identification of small molecules that inhibit (i) the interaction of FGF2 with Tec, (ii) tyrosine phosphorylation of FGF2 mediated by Tec in vitro and in a cellular context, and (iii) unconventional secretion of FGF2 from cells. We further demonstrate the specificity of these inhibitors for FGF2 because tyrosine phosphorylation of a different substrate of Tec is unaffected in their presence. Building on previous evidence using RNA interference, the identified compounds corroborate the role of Tec kinase in unconventional secretion of FGF2. In addition, they are valuable lead compounds with great potential for drug development aiming at the inhibition of FGF2-dependent tumor growth and metastasis.

Unconventional Secretion Mediates the Trans-cellular Spreading of Tau

The progressive deposition of misfolded hyperphosphorylated tau is a pathological hallmark of tauopathies, including Alzheimers disease. However, the underlying molecular mechanisms governing the intercellular spreading of tau species remain elusive. Here, we show that full-length soluble tau is unconventionally secreted by direct translocation across the plasma membrane. Increased secretion is favored by tau hyperphosphorylation, which provokes microtubule detachment and increases the availability of free protein inside cells. Using a series of binding assays, we show that free tau interacts with components enriched at the inner leaflet of the plasma membrane, finally leading to its translocation across the plasma membrane mediated by sulfated proteoglycans. We provide further evidence that secreted soluble tau species spread trans-cellularly and are sufficient for the induction of intracellular tau aggregation in adjacent cells. Our study demonstrates the mechanistic details of tau secretion and provides insights into the initiation and progression of tau pathology.

The molecular mechanism underlying unconventional secretion of fibroblast growth factor 2 from tumor cells

Fibroblast Growth Factor 2 (FGF2) is a potent cell survival factor involved in tumour‐induced angiogenesis. FGF2 is secreted from cells through an unconventional secretory mechanism based upon direct translocation across the plasma membrane. The molecular mechanism underlying this process depends on a surprisingly small set of trans‐acting factors that are physically associated with the plasma membrane. FGF2 membrane translocation is mediated by the ability of FGF2 to oligomerise and to insert into the plasma membrane in a PI(4,5)P2‐dependent manner. Membrane‐inserted FGF2 oligomers are dynamic translocation intermediates that are disassembled at the extracellular leaflet mediated by membrane proximal heparan sulphate proteoglycans. This process results in the exposure of FGF2 on cell surfaces as part of its unconventional mechanism of secretion. Although the trans‐acting factors and cis‐elements in FGF2 required for unconventional secretion have been known for a while, the core mechanism of this mysterious process has now been reconstituted with purified components establishing the molecular basis of FGF2 secretion from tumour cells.

Unconventional Protein Secretion: Fibroblast Growth Factor 2 and Interleukin-1β as Examples

N-terminal signal peptides are a hallmark of the vast majority of soluble secretory proteins that are transported along the endoplasmic reticulum (ER)/Golgi-dependent pathway. They are recognized by signal recognition particle (SRP), a process that initiates membrane translocation into the lumen of the ER followed by vesicular transport to the cell surface and release into the extracellular space. Beyond this well-established mechanism of protein secretion from eukaryotic cells, a number of extracellular proteins with critical physiological functions in immune surveillance and tissue organization are known to be secreted in an SRP-independent manner. Such processes have collectively been termed ‘unconventional protein secretion’ and, while known for more than two decades, their underlying mechanisms are only beginning to emerge. Different types of unconventional secretory mechanisms have been described. The aim of this article is to critically assess our current knowledge of this type of unconventional secretion focusing on fibroblast growth factor 2 (FGF2) and interleukin-1β as prototypical examples.

Die molekulare Entschlüsselung un kon - ventioneller Sekretionsmechanismen

The vast majority of secretory proteins is characterized by N-terminal signal peptides that allow for co-translational translocation into the lumen of the endoplasmic reticulum, the initial compartment of the classical ER/Golgi-dependent secretory pathway. However, extracellular proteins with fundamental physiological functions in immune surveillance and tissue organization have been identified that lack signal peptides. The molecular mechanisms by which these unconventional secretory proteins reach the extracellular space are beginning to emerge.